The present invention relates to the generation of hydrogen in cyanobacteria and isolated polynucleotides for same.
The development of a clean, sustainable and economically viable energy supply for the future is one of the most urgent challenges of our generation. Oil production is expected to peak in the near future and economically viable oil reserves are expected to be largely depleted by 2050. A viable hydrogen economy requires clean, sustainable and economic ways of generating hydrogen. Current hydrogen production depends almost entirely on the use of non-renewable resources (i.e. steam reformation of natural gas, coal gasification and nuclear power driven electrolysis of water). Although these approaches are initially likely to drive a transition towards a hydrogen economy, the hydrogen produced is more expensive and contains less energy than the non-renewable energy source from which it is derived. In addition, the use of fossil fuels and nuclear power is unsustainable. Therefore, there is a clear need to establish economically viable means of hydrogen production.
A particularly desirable option is the production of hydrogen using photosynthetic machinery, since the ultimate energy source is solar energy. The twin hearts of the photosynthetic machinery in plants, algae, and cyanobacteria are the two photochemical reaction centers known as Photosystem I (PSI) and Photosystem II (PSII). PSII drives the most highly oxidizing reaction known to occur in biology, splitting water into oxygen, protons and electrons. Oxygen is released into the atmosphere and is responsible for maintaining aerobic life on Earth. The derived electrons are passed along the photosynthetic electron transport chain from PSII via Plastoquinone (PQ) to Cytochrome b6f (cyt b6f) and Photosystem I (PSI). From PSI, most of the negative redox potential is stabilized in the form of reduced ferredoxin (Fd) that serves as an electron donor to ferredoxin-NADP+-reductase (FNR) enzyme. Under normal physiological conditions, Fd reduces NADP+ to NADPH via the Fd-FNR complex. In a parallel process (photophosphorylation), H+ are released into the thylakoid lumen where they generate a H+ gradient that is used to drive ATP production via ATP synthase. NADPH and ATP are subsequently used to produce starch and other forms of energy storage biomass.
Some green algae and cyanobacteria have evolved the ability to channel the protons and electrons stored in starch into hydrogen production under anaerobic conditions by expressing a hydrogenase enzyme. [Wunschiers, Stangier et al. 2001, Curr Microbiol 42(5): 353-60; Happe and Kaminski 2002, Eur J Biochem 269(3): 1022-32]. The hydrogenase enzyme is localized in the chloroplast stroma and obtains electrons from ferredoxin or flavodoxin that is reduced by Photosystem I and thus competes with FNR for the PSI generated electrons. However, oxygen is a powerful inhibitor of the hydrogenase enzyme and thus, the generation of hydrogen in these organisms is only transient. The most important challenge in photosynthetic hydrogen production is its spatial and/or temporal separation from oxygen production.
Efforts to generate oxygen-tolerant algal hydrogenases have not met with much success [Seibert et al. 2001, Strategies for improving oxygen tolerance of algal hydrogen production. Biohydrogen II. J. M. Miyake, T.; San Pietro, A., eds, Oxford, UK: Pergamon 67-77]. McTavish et al [J Bacteriol 177(14): 3960-4, 1995] have shown that site-directed mutagenesis of Azotobacter vinelandii hydrogenase can render hydrogen production insensitive to oxygen inhibition, but with a substantial (78%) loss of hydrogen evolution activity.
Plant and algal chloroplasts and cyanobacterial photosynthetic membranes contain two photosystems: PSII mediates the transfer of electrons from water (the initial electron donor) to the plastoquinone pool and PSI mediates electron transfer from plastocyanin to ferredoxin, thereby generating reducing power needed for CO2 fixation in the form of NADPH. While PSII is known to be sensitive to photodamage, PSI is considered to be more stable than PSII. Therefore, it is conceivable that preventing the assembly of PSII should result in its rapid inactivation under sunlight, and cessation of water splitting (and oxygen generation). Melis (U.S. Patent Application No. 2001/005343) teaches a process in which the inhibition of hydrogenase activity was lifted by temporally separating the oxygen generating water splitting reaction, catalyzed by PSII, from the oxygen sensitive hydrogen production catalyzed by the chloroplast Hydrogenase (HydA). This separation was achieved by culturing green algae first in the presence of sulfur to build stores of an endogenous substrate and then in the absence of sulfur. The removal of sulfur results in the inactivation of Photosystem II so that cellular respiration leads to anaerobiosis, the induction of hydrogenase, and sustained hydrogen evolution in the light.
The Melis process is, however, subject to considerable practical constraints. The actual rate of hydrogen gas accumulation is at best 15 to 20% of the photosynthetic capacity of the cells [Melis and Happe 2001, Plant Physiol. November; 127(3):740-8] and suffers the inherent limitation that hydrogen production by sulfur deprivation of the algae cannot be continued indefinitely. The yield begins to level off and declines after about 40-70 hours of sulfur deprivation. After about 100 hours of sulfur deprivation the algae need to revert to a phase of normal photosynthesis to replenish endogenous substrates.
International Publication No. WO 03/067213 describes a process for hydrogen production using Chlamydomonas reinhardtii wherein the algae has been genetically modified to down regulate expression of a sulfate permease, CrcpSulP, through the insertion of an antisense sequence. This is said to render obsolete prior art sulfur deprivation techniques, as it obviates the need to physically remove sulfur nutrients from growth media in order to induce hydrogen production. The reduced sulfur uptake by the cell using this technique not only results in a substantial lowering of the levels of the major chloroplast proteins such as Rubisco, D1 and the LHCII, but also deprives the cell of sulfur for use in the biosynthesis of other proteins.
Ihara et al (Ihara, Nakamoto et al. 2006; Ihara, Nishihara et al. 2006) teach a fusion protein comprising membrane bound [NiFe] hydrogenase (from the β-proteobacterium Ralstonia eutropha H16) and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus as a direct light-to-hydrogen conversion system. The isolated hydrogenase-PSI isolated complex displayed light-driven hydrogen production at a rate of [0.58 μmol H2]/[mg chlorophyll] h in vitro. The inefficiency of this system is thought to be derived from the mismatched ability of the hydrogenase to accept electrons compared to the ability of PSI to donate electrons.
Peters et al [Science, 282, 4 Dec., 1998] teach the isolation of an Fe-only hydrogenase from clostridium pasteurianum which naturally comprises ferrodoxin-like structures. Although this hydrogenase is potentially capable of directly generating hydrogen under illuminated conditions, it is still inhibited by oxygen.
There is thus a widely recognized need for, and it would be highly advantageous to have, a sustainable and efficient process for photosynthetic hydrogen production devoid of the above limitations.